CN107006368A - The method for tissue culture of Queensland nut - Google Patents
The method for tissue culture of Queensland nut Download PDFInfo
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- CN107006368A CN107006368A CN201710287816.0A CN201710287816A CN107006368A CN 107006368 A CN107006368 A CN 107006368A CN 201710287816 A CN201710287816 A CN 201710287816A CN 107006368 A CN107006368 A CN 107006368A
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- queensland nut
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- 240000007575 Macadamia integrifolia Species 0.000 title claims abstract description 40
- 235000018330 Macadamia integrifolia Nutrition 0.000 title claims abstract description 40
- 235000003800 Macadamia tetraphylla Nutrition 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 25
- 239000007787 solid Substances 0.000 claims abstract description 46
- 238000005507 spraying Methods 0.000 claims abstract description 26
- 239000012530 fluid Substances 0.000 claims abstract description 16
- 235000015097 nutrients Nutrition 0.000 claims abstract description 16
- 238000000889 atomisation Methods 0.000 claims abstract description 13
- 238000005086 pumping Methods 0.000 claims abstract description 9
- UUTKICFRNVKFRG-WDSKDSINSA-N (4R)-3-[oxo-[(2S)-5-oxo-2-pyrrolidinyl]methyl]-4-thiazolidinecarboxylic acid Chemical compound OC(=O)[C@@H]1CSCN1C(=O)[C@H]1NC(=O)CC1 UUTKICFRNVKFRG-WDSKDSINSA-N 0.000 claims abstract description 6
- 239000002609 medium Substances 0.000 claims description 51
- 238000005286 illumination Methods 0.000 claims description 38
- 239000007788 liquid Substances 0.000 claims description 27
- 229930006000 Sucrose Natural products 0.000 claims description 23
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 23
- 239000005720 sucrose Substances 0.000 claims description 23
- 239000001963 growth medium Substances 0.000 claims description 17
- 235000013379 molasses Nutrition 0.000 claims description 17
- 229930191978 Gibberellin Natural products 0.000 claims description 16
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 claims description 16
- 239000003448 gibberellin Substances 0.000 claims description 16
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 claims description 15
- 229920001817 Agar Polymers 0.000 claims description 13
- 239000008272 agar Substances 0.000 claims description 13
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 10
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 10
- 239000004698 Polyethylene Substances 0.000 claims description 10
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 10
- 229960000367 inositol Drugs 0.000 claims description 10
- -1 polyethylene Polymers 0.000 claims description 10
- 229920000573 polyethylene Polymers 0.000 claims description 10
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 10
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 claims description 8
- 239000011159 matrix material Substances 0.000 claims description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- 238000005266 casting Methods 0.000 claims description 6
- 238000003860 storage Methods 0.000 claims description 6
- 238000012549 training Methods 0.000 claims description 6
- 238000009423 ventilation Methods 0.000 claims description 6
- RMOGWMIKYWRTKW-UONOGXRCSA-N (S,S)-paclobutrazol Chemical compound C([C@@H]([C@@H](O)C(C)(C)C)N1N=CN=C1)C1=CC=C(Cl)C=C1 RMOGWMIKYWRTKW-UONOGXRCSA-N 0.000 claims description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 5
- 239000005985 Paclobutrazol Substances 0.000 claims description 5
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 5
- 239000004327 boric acid Substances 0.000 claims description 5
- 239000000428 dust Substances 0.000 claims description 5
- 229960002523 mercuric chloride Drugs 0.000 claims description 5
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 5
- 229960003512 nicotinic acid Drugs 0.000 claims description 5
- 235000001968 nicotinic acid Nutrition 0.000 claims description 5
- 239000011664 nicotinic acid Substances 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- 238000007670 refining Methods 0.000 claims description 5
- 235000015393 sodium molybdate Nutrition 0.000 claims description 5
- 239000011684 sodium molybdate Substances 0.000 claims description 5
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 229960000344 thiamine hydrochloride Drugs 0.000 claims description 5
- 235000019190 thiamine hydrochloride Nutrition 0.000 claims description 5
- 239000011747 thiamine hydrochloride Substances 0.000 claims description 5
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
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- 230000015572 biosynthetic process Effects 0.000 claims description 4
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- 229960001484 edetic acid Drugs 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims 1
- 238000004659 sterilization and disinfection Methods 0.000 claims 1
- 230000035784 germination Effects 0.000 abstract description 3
- 230000035755 proliferation Effects 0.000 abstract description 3
- 239000005556 hormone Substances 0.000 abstract description 2
- 229940088597 hormone Drugs 0.000 abstract description 2
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- YGGXZTQSGNFKPJ-UHFFFAOYSA-N methyl 2-naphthalen-1-ylacetate Chemical compound C1=CC=C2C(CC(=O)OC)=CC=CC2=C1 YGGXZTQSGNFKPJ-UHFFFAOYSA-N 0.000 description 4
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
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- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 235000014103 egg white Nutrition 0.000 description 2
- 210000000969 egg white Anatomy 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
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- 241001070941 Castanea Species 0.000 description 1
- 235000014036 Castanea Nutrition 0.000 description 1
- 240000001008 Dimocarpus longan Species 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- 235000000235 Euphoria longan Nutrition 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 235000000292 Gouania lupuloides Nutrition 0.000 description 1
- 244000299452 Gouania lupuloides Species 0.000 description 1
- 240000003473 Grevillea banksii Species 0.000 description 1
- 235000010247 Heritiera littoralis Nutrition 0.000 description 1
- 241000208467 Macadamia Species 0.000 description 1
- 206010035148 Plague Diseases 0.000 description 1
- 241000208465 Proteaceae Species 0.000 description 1
- 241000534509 Telopea Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
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- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
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- 239000000463 material Substances 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 239000002420 orchard Substances 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of method for tissue culture of Queensland nut, comprise the following steps：Step 1: clip Queensland nut cuts the immature leaf of tip base portion, takes the leaf with bud in axil to make explant into the fruitful branch pumping branch then of annual bearer；Step 2: the first solid medium of access carries out Initial culture 10 days；Step 3: the second solid medium of access carries out squamous subculture 60 days；Step 4: the 3rd solid medium of access carries out culture of rootage 30 days；Step 5: the 4th solid medium of access carries out culture of rootage 30 days again；Gold-tinted fluorescent tube and ultraviolet lamp tube are set in tissue culture case, the fluid nutrient medium of during which spraying atomization.From fruitful branch, leaf of the pumping branch with bud makees explant progress tissue cultures to the present invention then, the hormone and nutriment of various concentrations are added in the medium, coordinate appropriate LED/light source irradiation, improve germination rate, proliferation rate, sprout extended length and the rooting rate of Queensland nut explant.
Description
Technical field
The present invention relates to the tissue culture field of Queensland nut.It is more particularly related to a kind of tissue of Queensland nut
Cultural method.
Background technology
Queensland nut, also known as Queensland's chestnut, macadamia.Its benevolence is rich in unrighted acid, protein, vitamin
Deng being of high nutritive value, local flavor is very unique, have the title of " king of dry fruit ".Queensland nut market prospects are had an optimistic view of, ecological benefits,
Economic benefit tool is good, is that a kind of oil content is high, nutritious, unique flavor edible nut, is constantly in the international market
State that supply falls short of demand.
Tissue culture propagating has been applied to the quick breeding of many forests, orchard fruit and ornamental crop.Simultaneously as
Free of contamination plant can be produced by carrying out breeding using tissue cultures, can effectively be reduced vegetable plague and be propagated risk, therefore, tissue training
Support the security for also improving kind of mass transter.In Proteaceae other plant, the white Australia of looking glass tree, Grevillea banksii R. Br, Gao Shi is such as spent more
The existing related research report such as mountain longan, Telopea speciosissma.At present, set up be suitable for the method for tissue culture of Queensland nut compared with
It is few, taken root mainly due to the explant of Queensland nut in culture of rootage Jiangnan, therefore, carry out Queensland nut tissue cultures skill
The research of art, sets up a set of tissue culturing system for being suitable for Queensland nut, has to the stock breeding process for accelerating Queensland nut
It is significant.
The content of the invention
It is an object of the invention to solve at least the above, and provide the advantage that at least will be described later.
It is a still further object of the present invention to provide a kind of method for tissue culture of Queensland nut, it is from fruitful branch pumping then
Leaf of the branch with bud makees explant and carries out tissue cultures, and the hormone and nutriment of various concentrations are added in the medium, coordinates
Appropriate LED/light source irradiation, improves germination rate, proliferation rate, sprout extended length and the rooting rate of Queensland nut explant.
In order to realize that there is provided a kind of tissue cultures side of Queensland nut according to object of the present invention and further advantage
Method, comprises the following steps：
Step 1: clip Queensland nut cuts the development of tip base portion not into the fruitful branch pumping branch then of annual bearer
Complete leaf, takes the leaf with bud in axil to make explant, is soaked with 1g/L mercuric chloride and aseptic water washing is used after 3min, be repeated 5 times；
Step 2: the first solid medium of access carries out Initial culture 10 days, cultivation temperature is 25 DEG C, illumination 12h/ days,
Wherein, natural lighting 6h, intensity of illumination 2000lux, artificial lighting 6h, pharosage are 80 μm of ol/m2S, the first solid
Culture medium is 1/2MS, 7g/L agar, 15g/L sucrose, 2g/L carbon dust, 15g/L molasses fermented liquid, 15mg/L egg
White peptone, pH is 5.8；
Step 3: the second solid medium of access carries out squamous subculture 60 days, cultivation temperature is 25 DEG C, illumination 12h/ days,
Wherein, natural lighting 5h, intensity of illumination 2000lux, artificial lighting 7h, pharosage are 80 μm of ol/m2S, the second solid
Culture medium is 1/2MS, 2mg/L 6- benzyls aminoadenine, 10mg/L gibberellin, 15g/L agar, 15g/L sucrose,
15g/L molasses fermented liquid, 2g/L inositol, 1mg/L nicotinic acid, 5mg/L paclobutrazol, pH is 5.8；
Step 4: the 3rd solid medium of access carries out culture of rootage 30 days, cultivation temperature is 25 DEG C, illumination 12h/ days,
Wherein, natural lighting 4h, intensity of illumination 2000lux, artificial lighting 8h, pharosage are 80 μm of ol/m2S, the 3rd solid
Culture medium is 1/2MS, 2mg/L 6- benzyls aminoadenine, 1.0mg/L gibberellin, 2mg/L indolebutyric acid, 15g/L fine jade
Fat, 15g/L sucrose, 15g/L molasses fermented liquid, 1mg/L thiamine hydrochloride, 2g/L inositol, 1mg/L sodium molybdate,
5mg/L boric acid, pH is 5.8；
Step 5: the 4th solid medium of access carries out culture of rootage 30 days again, cultivation temperature is 25 DEG C, illumination 12h/
My god, wherein, natural lighting 3h, intensity of illumination 2000lux, artificial lighting 9h, pharosage are 80 μm of ol/m2S, the 4th consolidates
Body culture medium is 1/2QL, 2mg/L 6- benzyls aminoadenine, 1.0mg/L gibberellin, 2mg/L indolebutyric acid, 5mg/L
Methyl α-naphthyl acetate, 15g/L sucrose, 15g/L molasses fermented liquid, 2mg/L puridoxine hydrochloride, 80mg/L ethylenediamine tetra-acetic acid two
Sodium, pH is 5.8；
Wherein, step 2~five are carried out in tissue culture case, the gold-tinted fluorescent tube and ultraviolet light of the quantity such as setting in tissue culture case
Pipe, the wavelength of gold-tinted fluorescent tube is 580nm, and the wavelength of ultraviolet lamp tube is 410nm, during the artificial lighting of step 2, gold-tinted fluorescent tube with
The startup quantity ratio of ultraviolet lamp tube is 1:1, the fluid nutrient medium of during which spraying atomization, spraying 10min, pause 5min repeat 4
Secondary, during the artificial lighting of step 3, the startup quantity ratio of gold-tinted fluorescent tube and ultraviolet lamp tube is 5:7, the liquid of during which spraying atomization
Culture medium, spraying 15min, pause 5min, is repeated 6 times, during the artificial lighting of step 4, the startup of gold-tinted fluorescent tube and ultraviolet lamp tube
Quantity ratio is 1:2, during which spraying atomization fluid nutrient medium, spraying 20min, pause 10min, be repeated 6 times, step 5 it is artificial
During illumination, the startup quantity ratio of gold-tinted fluorescent tube and ultraviolet lamp tube is 1:3, the fluid nutrient medium of during which spraying atomization, spraying
30min, pause 10min, are repeated 6 times, and the fluid nutrient medium is 1/2MS, 20g/L sucrose.
Preferably, the method for tissue culture of described Queensland nut, in addition to：
Step 6: rooted plantlet is placed in into 25 DEG C of lower refining seedlings 7 days, the culture medium of seedling root is removed, is transplanted to sterilizing
In the casting bed matrix of reason, and bedding layer of polyethylene film at an upper portion thereof, and raised every 5 days polyethylene film ventilation 5h.
Preferably, the method for tissue culture of described Queensland nut, the tissue culture case includes：
Casing, it is square frame structure that it, which includes four root posts and the cross section of eight spreader formation,；
Lid, it is hinged on a spreader of the casing, forms the lid that can close or open wide, and the lid is towards institute
State in atomizer and liquid storage box of the surface provided with UNICOM of box house, the liquid storage box and store fluid nutrient medium；
Four culture plates, it is hinged on the same root post of the casing from top to bottom, the height of the culture plate with
The height ratio of the column is 1:4, the cross section of the culture plate is overlapped with the cross section projection of the casing, the culture plate
Side wall on be evenly distributed with gold-tinted fluorescent tube and ultraviolet lamp tube, and one pair of which side wall and relative be provided with strip louvre, institute
State the bottom of culture plate and be provided with multiple culture dishes in the form of multiple row and columns, the row and columns of three culture plates above it
Between be equipped with air-vent, and size is sequentially reduced from top to bottom.
Preferably, the method for tissue culture of described Queensland nut, the culture plate is provided with 3 × 3 culture dish, institute
Air-vent is stated for groined type, the ratio of the width of three culture plate air-vents above from top to bottom is 3:2:1.
Preferably, the method for tissue culture of described Queensland nut, the outside of the top edge of the heat emission hole is to downward
A baffle plate is stretched out, the vertical projection of the baffle plate covers the vertical projection of the heat emission hole.
Preferably, the method for tissue culture of described Queensland nut, the culture dish is up big and down small structure.
The present invention at least includes following beneficial effect：
Firstth, by selection result branch, leaf of the pumping branch with bud makees explant, Initial culture, subculture training to the present invention then
The 1/2MS culture mediums using improvement are supported, culture of rootage is using root induction and a large amount of two stage cultures of taking root, using the 1/ of improvement
Obtain substantial amounts of aseptic seedling in 2MS culture mediums and 1/2QL culture mediums, cooperated with LED light source illumination condition, short time, proliferation rate and
Rooting rate is significantly improved, and average root length and mean elements are dramatically increased, and has broken the limit that weather and soil are bred to Queensland nut
System, survival rate is high after transplanting, effectively solves the problem of cuttage root system is undeveloped, graft survival rate is low；
Secondth, the first solid medium of the invention adds carbon dust and provides carbon source, molasses fermented liquid supplement sugar and peptone
Organic nitrogen is supplemented, closely knit adventitious bud callus is formed, the second solid medium adds inositol, nicotinic acid supplement organic matter, few
Measure paclobutrazol with gibberellin to coordinate, promote, to the induction of callus, growth, differentiation, clump to be sprouted in seedling base portion callus
Raw seedling, the 3rd solid medium adds thiamine hydrochloride, inositol supplement organic matter, and sodium molybdate, boric acid microelement-supplementing are small
Seedling base portion formation root restriction, the 4th solid medium adds puridoxine hydrochloride supplement organic matter, disodium ethylene diamine tetraacetate supplement
White root system is grown on a great number of elements, root restriction；
3rd, the present invention breaks the normal procedure using 610~720nm light radiations, using be suitable for Queensland nut growth can
It is gold-tinted and purple light to absorb light, by adjusting quantity ratio and then regulating illumination intensity, and cold light source does not produce heat, and reduction matrix is steamed
Rate of drying is sent out, promotes the differentiation speed of different phase, promote growing way；
4th, the present invention provides a kind of tissue culture case suitable for Queensland nut tissue cultures, with reference to it to the special of illumination
Demand is set in gold-tinted fluorescent tube and ultraviolet lamp tube, During Illumination by increasing atomizer on lid, setting saturating on culture plate
The spraying atmosphere of stomata culture medium for liquid is passed through, and the natural lighting stage turns to four incubators the position not covered mutually completely
Put and cultivated, four incubators are turned to the position covered up and down and carry out relative closure culture, radiating by the artificial lighting stage
Hole carries out cross-ventilation, promotes the differentiation and growth in each stage.
Further advantage, target and the feature of the present invention embodies part by following explanation, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Brief description of the drawings
Fig. 1 is structural representation of the invention；
The structural representation of tissue culture case when Fig. 2 is present invention progress natural lighting.
Embodiment
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to specification text
Word can be implemented according to this.
It should be noted that experimental method described in following embodiments, is conventional method unless otherwise specified, institute
Reagent and material are stated, unless otherwise specified, is commercially obtained；In the description of the invention, it is necessary to which explanation, is removed
Non- separately to have clearly regulation and limit, term " installation ", " connected ", " setting " should be interpreted broadly, for example, it may be stationary phase
Even, set or be detachably connected, set, or being integrally connected, set.For one of ordinary skill in the art
Speech, can understand the concrete meaning of above-mentioned term in the present invention with concrete condition.Term " transverse direction ", " longitudinal direction ", " on ", " under ",
The orientation or position relationship of the instruction such as "front", "rear", "left", "right", " vertical ", " level ", " top ", " bottom ", " interior ", " outer " are
Based on orientation shown in the drawings or position relationship, it is for only for ease of the description present invention and simplifies description, is not to indicate or dark
Specific orientation must be had, with specific azimuth configuration and operation by showing the device or element of meaning, therefore it is not intended that right
The limitation of the present invention.
<Embodiment 1>
A kind of method for tissue culture of Queensland nut, comprises the following steps：
Step 1: clip Queensland nut cuts the development of tip base portion not into the fruitful branch pumping branch then of annual bearer
Complete leaf, takes the leaf with bud in axil to make explant, is soaked with 1g/L mercuric chloride and aseptic water washing is used after 3min, be repeated 5 times；
Step 2: the first solid medium of access carries out Initial culture 10 days, cultivation temperature is 25 DEG C, illumination 12h/ days,
Wherein, natural lighting 6h, intensity of illumination 2000lux, artificial lighting 6h, pharosage are 80 μm of ol/m2S, the first solid
Culture medium is 1/2MS, 7g/L agar, 15g/L sucrose, 2g/L carbon dust, 15g/L molasses fermented liquid, 15mg/L egg
White peptone, pH is 5.8；
Step 3: the second solid medium of access carries out squamous subculture 60 days, cultivation temperature is 25 DEG C, illumination 12h/ days,
Wherein, natural lighting 5h, intensity of illumination 2000lux, artificial lighting 7h, pharosage are 80 μm of ol/m2S, the second solid
Culture medium is 1/2MS, 2mg/L 6- benzyls aminoadenine, 10mg/L gibberellin, 15g/L agar, 15g/L sucrose,
15g/L molasses fermented liquid, 2g/L inositol, 1mg/L nicotinic acid, 5mg/L paclobutrazol, pH is 5.8；
Step 4: the 3rd solid medium of access carries out culture of rootage 30 days, cultivation temperature is 25 DEG C, illumination 12h/ days,
Wherein, natural lighting 4h, intensity of illumination 2000lux, artificial lighting 8h, pharosage are 80 μm of ol/m2S, the 3rd solid
Culture medium is 1/2MS, 2mg/L 6- benzyls aminoadenine, 1.0mg/L gibberellin, 2mg/L indolebutyric acid, 15g/L fine jade
Fat, 15g/L sucrose, 15g/L molasses fermented liquid, 1mg/L thiamine hydrochloride, 2g/L inositol, 1mg/L sodium molybdate,
5mg/L boric acid, pH is 5.8；
Step 5: the 4th solid medium of access carries out culture of rootage 30 days again, cultivation temperature is 25 DEG C, illumination 12h/
My god, wherein, natural lighting 3h, intensity of illumination 2000lux, artificial lighting 9h, pharosage are 80 μm of ol/m2S, the 4th consolidates
Body culture medium is 1/2QL, 2mg/L 6- benzyls aminoadenine, 1.0mg/L gibberellin, 2mg/L indolebutyric acid, 5mg/L
Methyl α-naphthyl acetate, 15g/L sucrose, 15g/L molasses fermented liquid, 2mg/L puridoxine hydrochloride, 80mg/L ethylenediamine tetra-acetic acid two
Sodium, pH is 5.8；
Wherein, step 2~five are carried out in tissue culture case, the gold-tinted fluorescent tube and ultraviolet light of the quantity such as setting in tissue culture case
Pipe, the wavelength of gold-tinted fluorescent tube is 580nm, and the wavelength of ultraviolet lamp tube is 410nm, during the artificial lighting of step 2, gold-tinted fluorescent tube with
The startup quantity ratio of ultraviolet lamp tube is 1:1, the fluid nutrient medium of during which spraying atomization, spraying 10min, pause 5min repeat 4
Secondary, during the artificial lighting of step 3, the startup quantity ratio of gold-tinted fluorescent tube and ultraviolet lamp tube is 5:7, the liquid of during which spraying atomization
Culture medium, spraying 15min, pause 5min, is repeated 6 times, during the artificial lighting of step 4, the startup of gold-tinted fluorescent tube and ultraviolet lamp tube
Quantity ratio is 1:2, during which spraying atomization fluid nutrient medium, spraying 20min, pause 10min, be repeated 6 times, step 5 it is artificial
During illumination, the startup quantity ratio of gold-tinted fluorescent tube and ultraviolet lamp tube is 1:3, the fluid nutrient medium of during which spraying atomization, spraying
30min, pause 10min, are repeated 6 times, and the fluid nutrient medium is 1/2MS, 20g/L sucrose；
As shown in Figure 1, 2, the tissue culture case includes：
Casing 1, it is square frame structure that it, which includes four root posts and the cross section of eight spreader formation,；
Lid 2, it is hinged on a spreader of the casing 1, forms the lid that can be closed or open wide, the court of lid 2
It is provided with to the surface inside the casing 1 in the atomizer 21 and liquid storage box of UNICOM, the liquid storage box and stores liquid training
Support base；
Four culture plates 3, it is hinged on the same root post of the casing 1 from top to bottom, the height of the culture plate 3
Height ratio with the column is 1:4, the cross section of the culture plate 3 is overlapped with the cross section projection of the casing 1, the training
It is evenly distributed with the side wall for supporting disk 3 on gold-tinted fluorescent tube 31 and ultraviolet lamp tube 32, and one pair of which side wall relative provided with strip
Heat emission hole 33, the bottom of the culture plate 3 is provided with multiple culture dishes 34, three trainings above in the form of multiple row and columns
Air-vent 35 is equipped between the row and column for supporting disk 3, and size is sequentially reduced from top to bottom；
Wherein, the culture plate 3 is provided with 3 × 3 culture dish 34, and the air-vent 35 is groined type, above
The ratio of the width of three air-vents 35 of culture plate 3 from top to bottom is 3:2:1；The outside of the top edge of the heat emission hole 33 is downward
Extend a baffle plate, the vertical projection of the baffle plate covers the vertical projection of the heat emission hole 33；The culture dish 34 is upper big
Under small structure；
Step 6: rooted plantlet is placed in into 25 DEG C of lower refining seedlings 7 days, the culture medium of seedling root is removed, is transplanted to sterilizing
In the casting bed matrix of reason, and bedding layer of polyethylene film at an upper portion thereof, and raised every 5 days polyethylene film ventilation 5h.
<Comparative example 1>
A kind of method for tissue culture of Queensland nut, comprises the following steps：
Step 1: clip Queensland nut cuts the development of tip base portion not into the fruitful branch pumping branch then of annual bearer
Complete leaf, takes the leaf with bud in axil to make explant, is soaked with 1g/L mercuric chloride and aseptic water washing is used after 3min, be repeated 5 times；
Step 2: the first solid medium of access carries out Initial culture 10 days, cultivation temperature is 25 DEG C, illumination 12h/ days,
First solid medium is 1/2MS, 7g/L agar, 30g/L sucrose, and pH is 5.8；
Step 3: the second solid medium of access carries out squamous subculture 60 days, cultivation temperature is 25 DEG C, illumination 12h/ days,
Second solid medium is 1/2MS, 2mg/L 6- benzyls aminoadenine, 10mg/L gibberellin, 15g/L agar, 30g/L
Sucrose, pH is 5.8；
Step 4: the 3rd solid medium of access carries out culture of rootage 30 days, cultivation temperature is 25 DEG C, illumination 12h/ days,
3rd solid medium is 1/2MS, 2mg/L 6- benzyls aminoadenine, 1.0mg/L gibberellin, 2mg/L indolebutyric acid,
15g/L agar, 30g/L sucrose, pH is 5.8；
Step 5: the 4th solid medium of access carries out culture of rootage 30 days again, cultivation temperature is 25 DEG C, illumination 12h/
My god, the 4th solid medium is 1/2QL, 2mg/L 6- benzyls aminoadenine, 1.0mg/L gibberellin, 2mg/L indoles fourth
Acid, 5mg/L methyl α-naphthyl acetate, 30g/L sucrose, pH is 5.8；
Step 6: rooted plantlet is placed in into 25 DEG C of lower refining seedlings 7 days, the culture medium of seedling root is removed, is transplanted to sterilizing
In the casting bed matrix of reason, and bedding layer of polyethylene film at an upper portion thereof, and raised every 5 days polyethylene film ventilation 5h.
Transplanting will be paid special attention to keep casting bed matrix and air humidity initial stage, typically need totally-enclosed management one week or so, root
It can progressively be divulged information at 20 days or so according to situation, and remove covering, when season in spring and autumn is transplanted, need to shaded 10 days or so；Winter
When season transplants, shade 20 days or so, but key is that control nursery bed temperature is just conducive to surviving between 15~25 DEG C, general transition
After transplanting two months, seedling is with regard to interchangeable pot transplanting.
<Comparative example 2>
A kind of method for tissue culture of Queensland nut, comprises the following steps：
Step 1: clip Queensland nut cuts the development of tip base portion not into the fruitful branch pumping branch then of annual bearer
Complete leaf, takes the leaf with bud in axil to make explant, is soaked with 1g/L mercuric chloride and aseptic water washing is used after 3min, be repeated 5 times；
Step 2: the first solid medium of access carries out Initial culture 10 days, cultivation temperature is 25 DEG C, illumination 12h/ days,
First solid medium is 1/2MS, 7g/L agar, 15g/L sucrose, 2g/L carbon dust, 15g/L molasses fermented liquid,
15mg/L peptone, pH is 5.8；
Step 3: the second solid medium of access carries out squamous subculture 60 days, cultivation temperature is 25 DEG C, illumination 12h/ days,
Second solid medium is 1/2MS, 2mg/L 6- benzyls aminoadenine, 10mg/L gibberellin, 15g/L agar, 15g/L
Sucrose, 15g/L molasses fermented liquid, 2g/L inositol, 1mg/L nicotinic acid, 5mg/L paclobutrazol, pH is 5.8；
Step 4: the 3rd solid medium of access carries out culture of rootage 30 days, cultivation temperature is 25 DEG C, illumination 12h/ days,
3rd solid medium is 1/2MS, 2mg/L 6- benzyls aminoadenine, 1.0mg/L gibberellin, 2mg/L indolebutyric acid,
15g/L agar, 15g/L sucrose, 15g/L molasses fermented liquid, 1mg/L thiamine hydrochloride, 2g/L inositol, 1mg/L
Sodium molybdate, 5mg/L boric acid, pH is 5.8；
Step 5: the 4th solid medium of access carries out culture of rootage 30 days again, cultivation temperature is 25 DEG C, illumination 12h/
My god, the 4th solid medium is 1/2QL, 2mg/L 6- benzyls aminoadenine, 1.0mg/L gibberellin, 2mg/L indoles fourth
Acid, 5mg/L methyl α-naphthyl acetate, 15g/L sucrose, 15g/L molasses fermented liquid, 2mg/L puridoxine hydrochloride, 80mg/L second two
Amine tetraacethyl disodium, pH is 5.8；
Step 6: rooted plantlet is placed in into 25 DEG C of lower refining seedlings 7 days, the culture medium of seedling root is removed, is transplanted to sterilizing
In the casting bed matrix of reason, and bedding layer of polyethylene film at an upper portion thereof, and raised every 5 days polyethylene film ventilation 5h.
<Comparative example 3>
A kind of method for tissue culture of Queensland nut, be the same as Example 1, unlike, 2cm length is intercepted in step one and contains bud
Stem section be explant.
Average sprout number that the germination rate of each method for tissue culture, each explant are produced, the average length of sprout, take root
Rate, the data such as number, average root length, height of seedling of averagely taking root are as shown in table 1.
Table 1
Number of devices and treatment scale described herein are the explanations for simplifying the present invention.To the present invention application,
Modifications and variations will be readily apparent to persons skilled in the art.
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, the present invention is not limited
In specific details and shown here as the legend with description.
Claims (6)
1. a kind of method for tissue culture of Queensland nut, it is characterised in that comprise the following steps：
Step 1: clip Queensland nut cuts the immature of tip base portion into the fruitful branch pumping branch then of annual bearer
Leaf, take the leaf with bud in axil to make explant, with 1g/L mercuric chloride soak 3min after use aseptic water washing, be repeated 5 times；
Step 2: the first solid medium of access carries out Initial culture 10 days, cultivation temperature is 25 DEG C, illumination 12h/ days, wherein,
Natural lighting 6h, intensity of illumination 2000lux, artificial lighting 6h, pharosage are 80 μm of ol/m2S, the first solid medium
For 1/2MS, 7g/L agar, 15g/L sucrose, 2g/L carbon dust, 15g/L molasses fermented liquid, 15mg/L peptone, pH
For 5.8；
Step 3: the second solid medium of access carries out squamous subculture 60 days, cultivation temperature is 25 DEG C, illumination 12h/ days, wherein,
Natural lighting 5h, intensity of illumination 2000lux, artificial lighting 7h, pharosage are 80 μm of ol/m2S, the second solid medium
For 1/2MS, 2mg/L 6- benzyls aminoadenine, 10mg/L gibberellin, 15g/L agar, 15g/L sucrose, 15g/L
Molasses fermented liquid, 2g/L inositol, 1mg/L nicotinic acid, 5mg/L paclobutrazol, pH is 5.8；
Step 4: the 3rd solid medium of access carries out culture of rootage 30 days, cultivation temperature is 25 DEG C, illumination 12h/ days, wherein,
Natural lighting 4h, intensity of illumination 2000lux, artificial lighting 8h, pharosage are 80 μm of ol/m2S, the 3rd solid medium
For 1/2MS, 2mg/L 6- benzyls aminoadenine, 1.0mg/L gibberellin, 2mg/L indolebutyric acid, 15g/L agar,
15g/L sucrose, 15g/L molasses fermented liquid, 1mg/L thiamine hydrochloride, 2g/L inositol, 1mg/L sodium molybdate, 5mg/
L boric acid, pH is 5.8；
Step 5: the 4th solid medium of access carries out culture of rootage 30 days again, cultivation temperature is 25 DEG C, illumination 12h/ days,
Wherein, natural lighting 3h, intensity of illumination 2000lux, artificial lighting 9h, pharosage are 80 μm of ol/m2S, the 4th solid
Culture medium is 1/2QL, 2mg/L 6- benzyls aminoadenine, 1.0mg/L gibberellin, 2mg/L indolebutyric acid, 5mg/L naphthalene
Acetic acid, 15g/L sucrose, 15g/L molasses fermented liquid, 2mg/L puridoxine hydrochloride, 80mg/L ethylenediamine tetra-acetic acid two
Sodium, pH is 5.8；
Wherein, step 2~five are carried out in tissue culture case, and the gold-tinted fluorescent tube and ultraviolet lamp tube of the quantity such as setting, yellow in tissue culture case
The wavelength of light lamp tube is 580nm, and the wavelength of ultraviolet lamp tube is 410nm, during the artificial lighting of step 2, gold-tinted fluorescent tube and ultraviolet light
The startup quantity ratio of pipe is 1:1, the fluid nutrient medium of during which spraying atomization, spraying 10min, pause 5min are repeated 4 times, step
During three artificial lighting, the startup quantity ratio of gold-tinted fluorescent tube and ultraviolet lamp tube is 5:7, the fluid nutrient medium of during which spraying atomization,
Spraying 15min, pause 5min, are repeated 6 times, during the artificial lighting of step 4, the startup quantity ratio of gold-tinted fluorescent tube and ultraviolet lamp tube
For 1:2, the fluid nutrient medium of during which spraying atomization, spraying 20min, pause 10min are repeated 6 times, the artificial lighting of step 5
When, the startup quantity ratio of gold-tinted fluorescent tube and ultraviolet lamp tube is 1:3, during which spraying atomization fluid nutrient medium, spraying 30min, temporarily
Stop 10min, be repeated 6 times, the fluid nutrient medium is 1/2MS, 20g/L sucrose.
2. the method for tissue culture of Queensland nut as claimed in claim 1, it is characterised in that also include：
Step 6: rooted plantlet is placed in into 25 DEG C of lower refining seedlings 7 days, the culture medium of seedling root is removed, is transplanted to sterilization treatment
In casting bed matrix, and bedding layer of polyethylene film at an upper portion thereof, and raised every 5 days polyethylene film ventilation 5h.
3. the method for tissue culture of Queensland nut as claimed in claim 1, it is characterised in that the tissue culture case includes：
Casing, it is square frame structure that it, which includes four root posts and the cross section of eight spreader formation,；
Lid, it is hinged on a spreader of the casing, forms the lid that can close or open wide, and the lid is towards the case
The surface in internal portion is provided with the atomizer and liquid storage box of UNICOM, the liquid storage box and stores fluid nutrient medium；
Four culture plates, it is hinged on the same root post of the casing from top to bottom, the height of the culture plate with it is described
The height ratio of column is 1:4, the cross section of the culture plate is overlapped with the cross section projection of the casing, the side of the culture plate
It is evenly distributed with wall in gold-tinted fluorescent tube and ultraviolet lamp tube, and one pair of which side wall relative provided with strip louvre, the training
The bottom for supporting disk is provided between multiple culture dishes, the row and column of three culture plates above in the form of multiple row and columns
Provided with air-vent, and from top to bottom, size is sequentially reduced.
4. the method for tissue culture of Queensland nut as claimed in claim 3, it is characterised in that the culture plate is provided with 3 × 3
Culture dish, the air-vent is groined type, and the ratio of the width of three culture plate air-vents above from top to bottom is
3:2:1。
5. the method for tissue culture of Queensland nut as claimed in claim 3, it is characterised in that the top edge of the heat emission hole
Outside extends downwardly out a baffle plate, and the vertical projection of the baffle plate covers the vertical projection of the heat emission hole.
6. the method for tissue culture of Queensland nut as claimed in claim 3, it is characterised in that the culture dish is up big and down small
Structure.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107494158A (en) * | 2017-09-28 | 2017-12-22 | 马山县荷松种植专业合作社 | Improve the engrafting method of Queensland nut fruit quality |
| CN107810730A (en) * | 2017-09-28 | 2018-03-20 | 马山县荷松种植专业合作社 | The engrafting method of Queensland nut |
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| CN204653244U (en) * | 2014-09-25 | 2015-09-23 | 武汉沃田生态科技有限公司 | A kind of plantlet in vitro outside sprout-cultivating-bottle incubator |
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Cited By (2)
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| CN107494158A (en) * | 2017-09-28 | 2017-12-22 | 马山县荷松种植专业合作社 | Improve the engrafting method of Queensland nut fruit quality |
| CN107810730A (en) * | 2017-09-28 | 2018-03-20 | 马山县荷松种植专业合作社 | The engrafting method of Queensland nut |
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